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An Inducible and Reversible Mouse Genetic Rescue System

Inducible and reversible regulation of gene expression is a powerful approach for uncovering gene function. We have established a general method to efficiently produce reversible and inducible gene knockout and rescue in mice. In this system, which we named iKO, the target gene can be turned on and off at will by treating the mice with doxycycline. This method combines two genetically modified mouse lines: a) a KO line with a tetracycline-dependent transactivator replacing the endogenous target gene, and b) a line with a tetracycline-inducible cDNA of the target gene inserted into a tightly regulated (TIGRE) genomic locus, which provides for low basal expression and high inducibility. Such a locus occurs infrequently in the genome and we have developed a method to easily introduce genes into the TIGRE site of mouse embryonic stem (ES) cells by recombinase-mediated insertion. Both KO and TIGRE lines have been engineered for high-throughput, large-scale and cost-effective production of iKO mice. As a proof of concept, we have created iKO mice in the apolipoprotein E (ApoE) gene, which allows for sensitive and quantitative phenotypic analyses. The results demonstrated reversible switching of ApoE transcription, plasma cholesterol levels, and atherosclerosis progression and regression. The iKO system shows stringent regulation and is a versatile genetic system that can easily incorporate other techniques and adapt to a wide range of applications.

Introduction
In the post-genome era, a major challenge is deciphering the function of thousands of newly identified genes. One of the main approaches for studying gene function involves inactivation of genes in cells or animals using random (chemical or insertional) mutagenesis or gene targeting. A common problem with these methods stems from the fact that the gene of interest is usually mutated throughout the animal's life. As a result, 1) in many cases the mutation leads to embryonic or neonatal lethality, precluding the assessment of the gene's function in later life; 2) in viable mutants interpretation of observed phenotypes is often complicated by the inability to distinguish the direct effects of the gene loss at the time of observation from the results of developmental abnormalities caused by the gene loss earlier in life; 3) in still other cases, life-long absence of a gene product causes compensatory adjustments of activities of other genes precluding the elucidation of the function of the gene of interest. Conditional knockout and gene expression technologies, such as the Cre/lox-mediated tissue-specific knockout [1] and the tetracycline (Tet) regulated transcriptional activation system [2], can regulate gene expression in a more spatially and temporally controlled fashion. However, these technologies are often laborious to establish and the results are frequently variable.
Here we report the development of a system that provides for the inducible and reversible gene inactivation in the mouse and can also be readily scaled up for high-throughput applications. The iKO system is a binary approach based on the Tet-dependent regulatory technology. It involves the combination of two mouse lines -a KO line that expresses the Tet-transactivator (tTA or rtTA) in place of the gene of interest, and a TIGRE (for tightly regulated) line that contains the gene of interest under the control of the Tet-responsive element (TRE) at a predetermined genomic locus. It has the advantage of, 1) ability to turn genes on or off at will by adding or removing doxycycline (Dox) at any time during the animal's life, thus minimizing embryonic lethality, developmental effects, and compensatory effects; 2) high degree of regulation to any gene inserted at the TIGRE locus, which has been selected to confer minimal basal expression and high inducibility, and to insert any gene of interest in a single step by Cre/loxP recombination; 3) efficiency; the design allowing streamlined production of both KO and TIGRE mice makes it possible to generate iKOs for a large number of genes in a costeffective manner; 4) flexibility; KO and TIGRE lines can be engineered independently and combined in numerous ways, making a wide range of applications possible.
As a proof of concept, we report the characterization of an iKO of the apolipoprotein E gene (ApoE iKO). ApoE plays a key role in regulating cholesterol metabolism and atherosclerosis progression. ApoE KO mice develop hypercholesterolemia and atherosclerosis that closely resemble the human conditions and are rapidly reversed when APOE protein is supplied [3,4,5,6,7,8]. Thus, inducible and reversible regulation of ApoE expression could result in rapid physiological changes, which in turn can help assess the iKO technology. Furthermore, the phenotype of ApoE deficiency is quantifiable and very sensitive to leaky expression, allowing for the evaluation of the stringency of gene regulation by iKO technology [9]. Here we demonstrate that in the ApoE iKO mice, ApoE gene expression, as well as blood cholesterol levels, is tightly controlled by Dox. In the presence of Dox, ApoE is expressed and the cholesterol levels are low; in its absence, the reverse is observed. Furthermore, on examination of aortic atherosclerosis in the ApoE iKO mice we found that Dox treatment before the onset of atherosclerotic lesions completely prevented lesion formation and Dox treatment after extensive lesions had already formed resulted in regression of the lesions. These results demonstrate the reversibility of the iKO, leading to phenotype switching within the same animal. ApoE iKO is also useful in its own right as a novel model system for the study of molecular mechanisms underlying atherosclerosis progression and regression.

Principle of the iKO System
As illustrated in Figure 1A, two genetically modified mouse strains are created. The first is a KO line in which a Tet-dependent transactivator (rtTA in this example) is inserted into the target gene (Gene X). The insertion inactivates Gene X, and places rtTA under the control of the endogenous promoter of Gene X. The KO line can be generated via either homologous recombination or insertional mutagenesis. The second line (TIGRE) contains an additional copy of Gene X cDNA (or genomic fragments) driven by TRE-promoter inserted in a specific locus in the genome (the TIGRE locus), which has been preselected for low basal transcriptional activity and high inducibility. When these two lines are crossed, rtTA protein produced from the KO allele can activate the TRE-Gene X in the TIGRE locus only in the presence of Dox. (Alternatively, tTA can be used, which works in the opposite way -Tet-off instead of Tet-on.) Figure 1B illustrates the breeding of KO and TIGRE lines to produce the iKO mouse, which is homozygous for the KO locus and carries one copy of the TIGRE allele. The status of the iKO mouse is regulated by Dox. In the absence of Dox, rtTA protein is produced, but is inactive. As a result, TRE-Gene X is silent, Gene X protein is not produced and the KO phenotype is manifested. In the presence of Dox, rtTA stimulates the synthesis of Gene X protein from the TIGRE locus in the same cells in which the endogenous gene would normally be expressed. Expression complements the missing endogenous Gene X activity and leads to phenotypically normal animals. Thus, one can switch between wild type and KO state of animals by simply adding or removing Dox (e.g. with food).

Selection of TIGRE Loci
To screen for TIGRE loci, we constructed a Moloney murine leukemia virus (MoMLV)-based retroviral vector, pRTonZ (Figure 2A), in which the TRE-controlled lacZ gene was used as a reporter for gene regulation. Retroviral transduction at low multiplicity of infection ensures integration of a single copy of the TRE-lacZ unit into the genome. pRTonZ contains a modified neomycin phosphotransferase gene, loxneo, in which initiating AUG has been placed upstream of a loxP site in frame with the neo coding region. Once optimal locus is selected, it is utilized as a target site for transgene-integration by the scheme shown in Figures 2B and 2C. The TRE-lacZ unit is removed by Cremediated recombination of flanking loxP sites, leaving one loxP site and the neo gene in the genome ( Figure 2B). Since the promoter of the loxneo gene as well as the initiating AUG are also removed, ES cells become G418-sensitive. In this configuration, any gene of interest can be introduced into the same locus by Cre/ loxP recombination ( Figure 2C). Recombinant ES clones can be selected by G418-resistance because the neo expression unit is reconstituted. PCR screening showed that .90% of these G418resistant clones had correct insertion of the new gene.
Optimal loci were initially screened in ES cells. The ES cell line used was derived from CJ7 [10], of 129/Sv background. Following infection with the pRTonZ retroviral vector, G418-resistant (G418 r ) clones were stained with X-gal ( Figure 3A). Most clones showed mosaic staining pattern. By the percentage of the X-galstained cell population, ES clones were classified into 4 categories ( Figure 3A): class I (,1% of X-gal-stained cells) to class IV (.50% of X-gal-stained cells). From 242 ES clones analyzed, 55 clones were classified into class I. Inducibility was examined by bgalactosidase (b-gal) activity after transfecting 43 class I clones with a tTA expression vector ( Figure 3B). We set the cut-off value for high induction level at 1500 munits/mg of protein and nine clones belonged to this category.
Those loci were further examined in mice. Three independent TRE-lacZ mouse lines were generated from class I ES clones T1, T2 and T3 ( Figure 3C). Heterozygous TRE-lacZ mice were

Author Summary
We describe a technology for the creation of inducible and reversible gene inactivation in mice. It combines two genetically modified mouse lines: a knock-out line with a tetracycline transactivator replacing the endogenous target gene, and a line in which a tetracycline-inducible cDNA of the target gene has been inserted into a specific genomic locus. A critical component of this system is the unique chromosomal loci we have identified and engineered that offer a platform for easy insertion of any gene of interest for tightly controlled expression. Because of its simple binary nature, allowing independent modification of each of the two components and possibility of use in a high-throughput mode, we believe that our system will be useful for multiple applications, such as introducing mutant or humanized form of the target gene as well as functional manipulating tools. We have applied this technology to the Apolipoprotein E (ApoE) gene and have demonstrated that: a) the expression of ApoE is strictly dependent on the presence of doxycycline, a tetracycline group antibiotic, in the mouse diet, b) in the absence of doxycycline (ApoE repressed) atherosclerotic plaques are formed, confirming the importance of ApoE in the process, and c) upon re-induction of ApoE in the animals with doxicyclin, atherosclerosis regressed.
crossed to MMTV-tTA mice, which has been reported to be transcriptionally active in a wide variety of cell types [11], allowing for the examination of lacZ induction in various tissues. Three genotypes of mice (lacZ(2)tTA(2), lacZ(+)tTA (2), and lacZ(+)tTA(+)) were analyzed for b-gal activity ( Figure 3C). Activity of lacZ(2)tTA(2) represents endogenous eukaryotic bgal activity. Difference between lacZ(2)tTA(2) and lacZ(+)tTA (2) indicates basal activity of lacZ gene in the absence of tTA, and comparison of lacZ(+)tTA(2) and lacZ(+)tTA(+) reveals induction levels in the presence of tTA. Overall, the three mouse lines showed similar pattern of b-gal activity, although some differences were also seen. Basal activities were low but detectable in many tissues, and they were comparable to the values measured in the parental ES clones. b-gal activity was inducible in almost every tissue, and overall induction levels correlated with the expression levels of tTA ( Figure 3C, bottom panel).

Improvement of Gene Regulation Control at the TIGRE Loci
Although the three loci T1, T2 and T3 showed tight regulation of gene expression, basal activity was still detectable in many tissues. This could result from enhancers in the vicinity of the integration sites. To solve this problem, we flanked the TRE-lacZ reporter by the insulator sequence derived from the chicken bglobin locus [12,13]. The insulator sequences were introduced into all three loci (T1, T2, T3) by Cre-mediated recombination according to the scheme shown in Figures 2B and 2C (see Figure  S1 for details) and regulation of the lacZ gene was examined by transient expression of tTA in ES cells ( Figure 4A). The insulators reduced basal b-gal activity to levels indistinguishable from wild type ES cells. In contrast, inducibility was not impaired by the insulator sequence, indicating its effectiveness for increasing stringency of gene regulation. The insulators were also introduced into class II, III and IV ES clones, which showed high basal activity ( Figure 3A). Although the insulator sequences were effective, basal activity was still clearly detectable in every clone ( Figure 4B). To test whether tight regulation is achieved by using other genes, we replaced the lacZ gene with a luciferase gene ( Figure S2). With this reporter, insulators reduced basal activity by 14, 20 and 11 fold in T1, T2 and T3 loci, respectively, bringing it close to the instrument detection limit ( Figure 4C). Calculation of the number of luciferase molecule per ES cell (Supporting Methods) revealed that on average only 1, 0.7 and 0.3 luciferase molecule were expressed per cell in T1, T2 and T3 loci, respectively ( Figure 4D). Importantly, induced levels were not impaired by the insulator (Figure 4C), leading to high induction ratio of luciferase activity. Similar basal activity levels could also be achieved by expressing transrepressor ( Figure S3). However, our system is simpler because no additional protein expression is required.
To evaluate the insulator effect in vivo, we also generated mice containing TRE-lacZ gene and insulators, or TRE-luciferase (Luc) gene and insulators at the TIGRE locus (T1), and bred them with mice containing tTA under the control of various promoters. Basal expression was examined in multiple tissues of TRE-Luc mice by RT-PCR ( Figure 4E). Luc mRNA was undetectable in all tissues examined except testis, indicating very low basal levels of expression throughout the body. To examine if the Luc transcript detected in testis can produce functional Luc proteins, we conducted luciferase activity assays from protein extracts of all these tissues. The luciferase activity in testis was at the similar low basal level as all other tissues examined (data not shown) as well as the original TIGRE ES clone containing TRE-Luc with insulators that had been shown to express approximately one Luc molecule per cell ( Figure 4D), suggesting that the RT-PCR band detected in testis resulted from aberrant transcription that did not generate functional protein. To examine induced expression, we used lines of mice with tTA under the control of two brain-specific promoters: a-CaMKII (calcium/calmodulin-dependent protein kinase II) [14] or NSE (neuron-specific enolase) [15]. Sagital sections of 50 mm thickness across the entire brain from TRE-lacZ, P CAMKII -tTA/TRE-lacZ or P NSE -tTA/TRE-lacZ mice were stained for b-galactosidase (gal) activity and representative sections are shown in Figure 4F. In TRE-lacZ mice, no b-gal staining was seen in any parts of the brain. In P CAMKII -tTA/TRE-lacZ or P NSE -tTA/TRE-lacZ mice, intense b-gal staining was observed in specific regions of the brain defined by the two promoters respectively. These results demonstrate tight control of the TIGRE locus in animals.

Characterization of the TIGRE Locus
Using the splinkerette PCR method [16], we obtained genomic fragments covering either 59 or 39 junctions of the TIGRE vector insertion site in the T1 ES cell line. After sequencing the fragments, we determined the precise integration site of the viral TIGRE vector in the T1 TIGRE locus, which is located on chromosome 9. Genomic sequences surrounding the T1 TIGRE locus are shown in Figure 5A. Characteristic of retrovirus insertions, four nucleotides immediately adjacent to the insertion were duplicated and the viral TIGRE vector was inserted exactly in between the duplication. BLAT search of the UCSC Mouse Genome Browser with genomic sequences surrounding T1 revealed the localization of T1 locus to chr9 qA3 ( Figure 5B). The insertion site is flanked by two genes: AB124611 and Carm1. The insertion site is located 39 to the hypothetical gene AB124611 with unknown function and undetermined polyA site. The insertion site is also ,1.5 kb upstream of the transcriptional start of Carm1. Carm1 is ubiquitously expressed and Carm12/2 mice are embryonic lethal [17]. However, we have not observed overt developmental or other abnormalities in heterozygous or homozygous T1 TIGRE reporter lines TRE-lacZ or TRE-Luc (both with insulators), indicating that the viral insertion did not disrupt the nearby Carm1 gene.

KO Lines Produced from an ES Cell Library Mutagenized by a Retroviral Vector
KO lines with target genes replaced by rtTA or tTA can be produced by any gene targeting or insertional mutagenic methods. To implement high-throughput production of iKO mice, we have utilized a large-scale insertional mutagenesis ES cell library developed in house for the KO production [18]. Figure 6A upper panel illustrates the structure of the retroviral vector used. In the  Three ES clones were designated as T1, T2 and T3 as shown in the figure, and were used to generate mice. (C) Gene regulation in mice generated from tightly regulated ES clones. Three mouse strains were established from ES clones T1, T2 and T3, and crossed to particular case of ApoE, the virus is inserted in the third intron. The vector contains splice acceptor, stop codons, polyA signal and transcriptional terminator to ensure gene inactivation, which we confirmed to be the case by showing that ,90% of isolated genespecific ES clones were null alleles [18]. (The remaining were mostly knock-downs, and in nearly all these cases the retrovirus was inserted into 59 UTR (exon or intron), suggesting that retroviral insertions upstream of the coding regions of genes should be avoided.) The vector also includes the rtTA gene immediately downstream of the splice acceptor, stop codons and internal ribosome entry site (IRES), so that rtTA protein can be synthesized from the ApoE-IRES-rtTA hybrid transcript.
MMTV-tTA mouse. Top three panels: b-gal activity was measured in the following three genotypes: nontransgenic mice (lacZ(2)tTA (2), open bars); mice with lacZ gene but without tTA (lacZ(+)tTA (2)  To examine if random insertion of the retroviral vector containing rtTA into a gene results in rtTA expression reflecting the expression patterns of the inactivated gene, we compared rtTA transcription with the transcription of the endogenous gene by RT-PCR in different tissues for 26 different G protein coupled receptor (GPCR) KO lines generated from the library. Heterozygous mice carrying one allele of the intact endogenous gene and one allele interrupted by the rtTA-bearing retroviral vector were used to prepare total RNA samples from different tissues and amplify gene-specific transcript and rtTA transcript from the same RNA preps for side-by-side comparison. Figure 6B shows three examples of such comparison for genes P2Y6, RE2 and LGR6 respectively. The retroviral vector was inserted into a different part of each of the three genes -a 59UTR intron of P2Y6, an intron within the coding region of RE2 and the 15 th coding exon of LGR6. In all three cases, rtTA expression profiles closely resemble those of the endogenous genes to be inactivated. Real-time qPCR for 16 tissues (Table S1) also showed high degree of correlation between rtTA and endogenous gene from tissue to tissue. Overall, out of the 26 lines examined, 19 lines showed good correlation between rtTA and the endogenous gene's expression (Table S2), i.e. rtTA is expressed in the tissues where the corresponding endogenous gene is expressed and the relative ratio across different tissues for each transcript also appears similar between rtTA and endogenous gene. In the remaining 7 lines in which rtTA was not expressed well, 5 lines had the retroviral vector inserted upstream LGR6. Heterozygous mice are used so that the endogenous transcripts from the WT allele and the rtTA-containing hybrid transcripts from the KO allele can be amplified from the same RNA preps. Each RT-PCR reaction (RT+) has an RT-control run simultaneously to exclude any possibility of genomic DNA contamination. (C) Comparison of three transcripts by RT-PCR from the same RNA prep of the liver, the major site of normal ApoE production, of the ApoE+/2;TRE-ApoE mice -endogenous ApoE transcript (endoApoE), TRE-ApoE transcript from the TIGRE locus (TRE-ApoE) and ApoE-rtTA hybrid transcript (endoApoE-rtTA). Mice were fed either with or without Dox. As expected, expression of endoApoE and rtTA were independent of Dox. However, expression of TRE-ApoE was strictly dependent on Dox -it was undetectable in its absence and significantly expressed in its presence, indicating high degree of ApoE regulation achieved in the mice. doi:10.1371/journal.pgen.1000069.g006 Consequently they were lumped together as the ''WT group''.) Plasma cholesterol levels were first measured in mice fed with normal food without Dox (2Dox) and both ApoE iKO and KO mice showed significantly higher cholesterol levels compared to WT group mice (p = 0.35 between iKO and KO, p,0.0001 between iKO and WT or between KO and WT, Student's t-test). The mice were then switched to Dox-containing food, and plasma cholesterol levels were measured again 4 days (+Dox 4d) and 7 days (+Dox 7d) later. Cholesterol level of ApoE iKO mice dropped to WT levels in less than 4 days while that of ApoE KO remained high (p,0.0001 between iKO and KO or between KO and WT, p = 0.86 between iKO and WT). Sometime later, some Dox-treated mice were switched back to normal food, and plasma cholesterol levels were measured again 4 days (2Dox 4d) and 7 days (2Dox 7d) after Dox withdrawal. Cholesterol level of ApoE iKO mice significantly elevated by day 4 (p,0.001 between iKO and WT, p,0.01 between iKO and KO) and approached pre-Dox treatment level by day 7 of the first (and usually fairly large) intron of each gene. It has been recognized that the first intron (especially if it is large) could contain essential transcriptional regulatory elements, and thus insertions in this region might disrupt the transcriptional regulation and so should be avoided. Otherwise, our data showed that in the majority of insertional sites, rtTA could be expressed through the endogenous promoter upon integration via the retroviral vector.

Tightly Regulated ApoE Expression and Blood Cholesterol Levels by Dox in the ApoE iKO Mice
To prove the concept of iKO system, we generated ApoE iKO mice by creating ApoE KO line and ApoE TIGRE line separately and breeding them together ( Figure 6A), and attempted to model human conditions such as hypercholesterolemia and atherosclerosis. ApoE KO line was created by screening the mutant ES cell library as mentioned above. ApoE TIGRE line was created by inserting a TRE-ApoE transgene, flanked with 4 copies of chicken b-globin insulators, into the T1 TIGRE locus. RT-PCR analysis of heterozygous mice (ApoE+/2; TRE-ApoE) showed that expression of TRE-ApoE was strictly dependent on Dox ( Figure 6C). Real-time qPCR using primers specific for the endogenous ApoE or TRE-ApoE (2 sets of primers for each gene) showed that endogenous ApoE mRNA level is very high (compared to 18s rRNA), and the TRE-ApoE mRNA level in the presence of Dox is ,6.7 fold lower than the endogenous ApoE (DCt = 2.7 between TRE-ApoE and endogenous ApoE), while in the absence of Dox TRE-ApoE is .55,000 fold lower than the endogenous ApoE (DCt. 15.8). This shows the TRE-ApoE induction by Dox is .8,000 fold.
Next, we carried out phenotypic analysis of homozygous ApoE iKO mice (ApoE2/2; TRE-ApoE), i.e. mice having both endogenous ApoE alleles inactivated and carrying TRE-ApoE in the TIGRE locus. We analyzed blood cholesterol levels in these mice in the absence and presence of Dox. Constitutive KO (i.e. ApoE2/2 without TRE-ApoE in the TIGRE locus) and WT group mice that were littermates of iKO were used as controls. As shown in Figure 7A, in the absence of Dox the iKO mice had high cholesterol levels similar to that of the KO mice; in the presence of Dox, the iKO showed normal cholesterol levels, demonstrating that inducible expression of ApoE can lead to the reversion of the KO phenotype of hypercholesterolemia. When Dox was withdrawn, the cholesterol levels in the iKO mice rose again. These on/off switches occurred rapidly, within a few days after Dox administration or withdrawal.

Dox-Regulated Atherosclerosis Progression and Regression in the ApoE iKO Mice
We examined the atherosclerotic lesion formation in the aortas of the ApoE iKO mice. As controls we used KO and WT group mice that were littermates of iKO. In the absence of ApoE protein, aortic atherosclerotic lesions start to form around 3-4 months of age and progress continually with time. One set of iKO, KO and WT group mice were treated with Dox-containing food throughout their life, and were compared with mice fed with normal food. Figure 7B shows the atherosclerotic lesions formed around the arch region of the aorta, as visualized by Sudan IV staining. By 7 months of age, extensive lesions had formed in KO mice, regardless of whether they were treated with Dox or not, whereas WT mice did not have any lesions in the absence ( Figure 7B) or presence (data not shown) of Dox. The iKO mice developed extensive lesions in the absence of Dox (similar to KO mice), whereas in the presence of Dox no lesions had formed.
We further investigated what happens if ApoE protein expression is turned on after the atherosclerotic lesions have already formed. ApoE iKO and KO mice of 5 months of age were switched from normal food to Dox-containing food for the next 4 months. Aortic atherosclerotic lesions were examined before (at 5 months) and after (at 9 months) the Dox treatment. As shown in Figure 7C, at 5 months of age, both iKO and KO had developed similar levels of lesions. After 4 months of Dox treatment, the lesions in KO mice continued to grow, whereas in the iKO mice, the lesions had regressed nearly completely with only scar-like tissues remaining, suggesting that the lipid-containing foam cells have disappeared from the lesions. The results were verified by quantification and statistical analysis of the lesions by one-way ANOVA followed by Neuman-Keul's post hoc test ( Figure 7D).

Discussion
We have developed a reversible and inducible rescue system for gene KO in mice and have applied this method into the ApoE gene. Our system complements existing inducible gene expression approaches and provides certain advantages. The tamoxifendependent Cre-ERT2 [19,20] recombination can drive inducible knockout of the endogenous gene, however, it is irreversible and the efficiency of tamoxifen inducibility throughout the body is yet to be demonstrated. The usefulness of the Tet-inducible system [21,22] is critically dependent on the tightness of transcription induction and suppression. When either the Tet-transactivator or the target gene is randomly integrated into the genome, they are subjects to positional effects [23]. It is often necessary to screen (p,0.0001 between iKO and WT, p = 0.13 between iKO and KO). ***P,0.001. (B) Atherosclerotic lesion progression. Aortas were stained with Sudan IV to visualize the lesions in red. The arch region of the aorta contains the most extensive areas of lesions and is shown here. A group of iKO, KO and WT mice were treated with Dox-containing food starting before the onset of lesions, and were compared with mice fed with normal food. At 7 months of age, aortas were dissected from these mice and lesions were examined. ApoE iKO mice showed extensive aortic lesions as the KO mice in the absence of Dox, and yet no lesions at all as the WT mice in the presence of Dox. (C) Atherosclerotic lesion regression. ApoE iKO and KO mice of 5 months of age were switched from normal food to Dox-containing food. Aortic lesions were examined before (at 5 months) and after (at 9 months) the Dox treatment. After 4 months of Dox treatment, the lesions in KO mice continued to grow, whereas in the iKO mice the lesions had regressed. (D) Quantification of the aortic atherosclerotic lesion areas in the arch region above the first intercostal artery, as expressed by the percentage of lesion areas versus the whole aortic area in this segment. All genotypes are matched with ages for different Dox treatment. Dox-treated groups are: iKO+Dox: Dox food started at 2-4 months of age (before the onset of atherosclerosis); iKO+Dox regression: Dox food started at 5-6 months of age (after the onset of atherosclerosis); KO+Dox: Dox food started at 2-4 months of age (before the onset of atherosclerosis). The results are compared using one-way ANOVA followed by Neuman-Keul's post hoc test. Both iKO+Dox and iKO+Dox regression groups had significantly reduced atherosclerotic areas compared to the remaining groups. *P,0.05, **P,0.01. The iKO mice without Dox, as well as KO mice either with or without Dox, all developed comparable areas of lesions (p.0.05 in all pair-wise comparisons). The iKO mice treated with Dox before the onset of atherosclerosis had nearly no lesions and were significantly different from the above groups (p,0.01 iKO+Dox versus iKO2Dox; p,0.01 iKO+Dox versus KO2Dox; p,0.05 iKO+Dox versus KO+Dox). The iKO mice treated with Dox after the onset of atherosclerosis had significantly reduced atherosclerotic areas compared to iKO mice without Dox or KO mice either with or without Dox (p,0.05 in all comparisons between iKO+Dox regression versus iKO2Dox, KO2Dox or KO+Dox). doi:10.1371/journal.pgen.1000069.g007 through multiple transgenic lines every time a new transgene is introduced. Attempts to target both tTA and TRE together into an endogenous gene locus to achieve inducible activation and inactivation were successful in a few cases [24,25,26], but this method is not applicable to most other genes as the TRE is easily subject to activation from nearby enhancers independent from tTA. Our iKO technique utilizes each gene's own promoter to direct the expression of transcriptional activators, e.g. rtTA and tTA. Therefore it is not limited to certain tissues and available tissue-specific promoter driven transgenic lines. It could be applicable to any gene in any tissue. Dox-regulated expression can be turned on and off rapidly, i.e. within a few days, and at any time in development or adult. It also allows analysis of the effects of gene inactivation in the same animal. The TIGRE locus has been selected to confer little or no basal transgene expression throughout the body while maintaining high inducibility, enabling stringent control of the on/off switching of the target gene. Our system also has the flexibility to allow for further improvements. For example, in some cases it may be more desirable to put the genomic copy of the gene under TRE promoter into the TIGRE locus instead of the cDNA to more precisely mimic the expression of the endogenous gene. Also, the IRES we have used to drive rtTA translation may not work uniformly well in all tissues, and can be replaced by other approaches such as using the viral 2Alike sequences for bicistronic translation [27] or direct targeting of tTA/rtTA into the ATG start codon of the endogenous gene.
Unique chromosomal loci for predicted gene expression provide fundamental tools for genetic studies. The most widely used is the ROSA26 locus [28] in which ubiquitous gene expression is achieved by endogenous promoter activity of this locus. The TIGRE loci identified in this study allow a different mode of gene regulation -they were selected from hundreds of insertion sites for tight gene regulation by an exogenous promoter. Therefore, the TIGRE loci offer a platform for easy insertion of any gene in a tightly regulated locus, applicable to not only the tetracycline system but also other gene expression systems utilizing exogenous promoters such as constitutively active promoters, tissue specific promoters, or other inducible promoters regulated by reagents such as ecdysone [29], mifepristone [30] and streptogramin [31]. Recently a similar approach was also applied to a human fibrosarcoma cell line to pre-screen optimal integration sites for transgenes [32]. In addition, here we demonstrated that the stringency of the regulation at TIGRE loci is further enhanced by the incorporation of insulators, i.e. basal expression level was reduced to less than one luciferase molecule per cell without impairing inducibility ( Figure 4C, D). Insulators were also shown previously to improve inducibility of randomly integrated TREreporters [33]. It should be noted that in our study the insulator effect was limited in many other loci ( Figure 4A versus 4B), demonstrating the uniqueness of the TIGRE loci. The genomic location of the T1 TIGRE locus, which was most extensively used in our study including the ApoE iKO mice, was determined ( Figure 5). Further manipulation of this locus would be possible to expand its application.
The stringent gene regulation is demonstrated in the ApoE iKO mice. It is known that regulation of blood cholesterol levels is very sensitive to the plasma ApoE protein levels. Even with the production of 3% of wild-type level of ApoE protein, the hypercholesterolemia and atherosclerosis phenotypes of the ApoE KO mice can be reversed [9]. Given this high sensitivity, the fact that our ApoE iKO mice in the uninduced state (i.e. in the absence of Dox) exhibit similarly high levels of cholesterol compared to ApoE KO mice indicates that there is hardly any expression of functional APOE. Reversing the KO at will is a particularly powerful approach in atherosclerotic regression studies. Doxtreatment of ApoE iKO mice results in expression of ApoE, marked reduction of plasma cholesterol levels, and regression of aortic atherosclerotic lesions. These findings are consistent with previous studies showing that aggressive lipid lowering or expression of ApoE can induce regression of pre-existing atherosclerotic lesion [7,8,34,35]. It is becoming increasingly clear that lesion regression is regulated by a complex interplay between lipids, inflammation and the immune system [35]. The ApoE iKO mice will allow detailed studies on the roles of specific genes in these complex interactions.
The binary nature of the iKO system is inherently simple, with the KO line serving dual roles: it could be used as a constitutive KO or combined with the TIGRE line to produce an inducible and reversible iKO. The 10-million clone ES cell library we utilized [18] has been estimated to contain insertional mutations for .90% of genes, and individual ES clones with retroviral insertions in a specific target gene can be rapidly identified through a PCR pooling strategy and subsequently isolated in a streamlined process. The identification and modification of the TIGRE locus allows rapid insertion of any gene of interest via co-transfection with Cre. Therefore, each component of the binary system, the KO or the TIGRE line, is amenable for high-throughput production to generate inducible and reversible KOs for a large number of genes.
It should be noted that the iKO system may not be applicable in certain situations where highly stringent gene regulation is required. For example, even though the system had enough stringency in low basal activity and high induction, the induced gene expression level is usually not exactly the same as the endogenous gene's level and this could be a problem for genes that are highly sensitive to gene dosage effect or show haploid deficiency. The kinetics of the system (days) may be also too slow for some developmental problems where transient expression of developmental genes are critical, although it should be noted that it could still work well in carefully thought-out developmental studies (as in [24]). In addition, it has been reported that rtTA often can not induce sufficient gene expression in the brain, at least partially due to developmental inactivation of the TRE promoter in neurons [36]. It appears that the tTA (Tet-off) system is better suited for the use in brain [14], as substantial b-gal induction was observed in brain with tTA ( Figure 4F).
Our results of the ApoE iKO mice and the quantitative data using lacZ and luciferase reporters suggest that the iKO system could be a useful tool in addressing a variety of biological questions. The two components of the iKO system can be independently modified, and pairing of their different forms can generate numerous combinations. In KO lines, genes with unique expression patterns are tagged with a transcription transactivator, which can control, in an inducible fashion, the expression of a variety of genes derived from TIGRE lines, enabling a number of additional applications in specific types of tissues or cells. Those include: 1) introducing mutant forms of the target gene into the TIGRE locus for better functional probing of different domains, splice variants, post-transcriptional modifications (e.g. phosphorylation), or modeling human diseases; 2) humanizing target genes by placing a human ortholog of the mouse gene under TRE control, which can facilitate drug efficacy studies; 3) placing a cytotoxic gene under TRE control to allow inducible cell-type specific ablation; 4) introducing a marker gene such as GFP, protein interacting probe, or transneuronal tracer, into the TIGRE locus for cell-type specific tagging, functional analysis, isolation of specific cell population, or mapping neuronal networks; 5) combining with recently developed RNAi techniques [37,38,39] to down-regulate any genes of interest in a tissue-specific and inducible manner; 6) re-engineering the TIGRE locus to place a TRE-driven Cre, and combining it with a tissue-specific rtTA or tTA line and floxed target genes to achieve inducible region-specific gene knockout [40].

Materials and Methods
See Text S1 online for a more detailed description of the methods.

Screening and Isolation of ES Clones with Insertions in Target Genes
Construction of ES cell library infected with the retrovirus and screening and isolation of ES clones with genes of interest inactivated by the viral insertion is described previously [18]. Specifically for ApoE, insertions were found by nested PCR analysis of the ES DNA using two vector-specific and gene-specific primer pairs. The ApoEspecific primers are antisense and located in the fourth exon. Several independent retroviral insertions in the ApoE gene were identified. PCR fragments were sequenced to confirm insertion into the gene. A library tube with a clone of interest identified by the PCR also contains a few hundred other ES cell clones. The sole desired clone was isolated from the mixture by three rounds of cell sorting and growing followed by PCR using the same pair of primers to identify positive clones. Additional PCRs using ApoE primers located in the third intron flanking the viral insertion site were conducted to confirm the precision of the insertion and integrity of the genomic sequence of ApoE.

Generation of TIGRE ES Clones Containing Target Genes
Full length cDNAs for the coding sequences of target genes were cloned into the TIGRE-targeting vector containing TRE, insulators, a PGK promoter and a pair of loxP sites ( Figure 5A lower panel). The TIGRE-targeting vectors were co-transfected with a Creexpressing plasmid into the neo-sensitive ES cells carrying the minimal TIGRE locus with a single loxP site and the promoterless, ATGless loxneo marker. When the TIGRE-targeting vector is integrated into the TIGRE locus through Cre/lox-mediated recombination, neo-resistance is restored to the ES cells by the addition of PGK promoter and in-frame fusion of ATG to the loxneo marker. Correctly integrated ES clones were identified and confirmed by PCR screening and southern blot analysis.

Animal Production and Maintenance
ES cell clones were injected into blastocysts of C57BL/6J mice following standard techniques. Chimeric mice were bred with C57BL/6J mice to test germline transmission and generate heterozygous mice. Mice from the corresponding KO lines and TIGRE lines were crossed with each other to produce inducible KO mice according to the scheme shown in Figure 1B. All mice used for studies in this paper were in a mixed genetic background of 50% 129S1/SvImJ and 50% C57BL/6. For the ApoE KO line, southern blot using rtTA coding sequence as probe confirmed the correct insertion of the retroviral vector into the endogenous ApoE gene. It also revealed an additional retroviral insertion somewhere else in the genome. The additional insertion was selectively bred out, and the ApoE iKO colony was maintained with a single retroviral insertion at the ApoE locus. MMTV-tTA, P CAMKII -tTA and P NSE -tTA mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All experimental procedures were approved by the Institutional Animal Care and Use Committee of NCI and Nura, Inc. in accordance with NIH guidelines.
Dox was administered to the mice through Dox-containing food, which was custom made by Bio-Serv (Frenchtown, NJ) to contain 2 g Dox per kilogram of food. The nutrition content of the Dox food (e.g. 19% protein and 8.6% fat) was very similar to the regular diet (20% protein, 9% fat) used in our colony. Therefore the switching of food types did not result in change of cholesterol levels in mice. Figure S1 Cre-mediated introduction of the insulator sequence into the LacZ gene at the TIGRE loci.


Meuffelsia, a New Genus of Long-Legged Flies from South Africa, with a Key to Afrotropical Peloropeodine and Allied Genera (Diptera: Dolichopodidae)

ABSTRACT The genus Meuffelsia Grichanov, gen. n. is described from South Africa to accommodate two new species, M. erasmusorum Grichanov. sp. n. and M. manningi Grichanov, sp. n. The new genus has been placed in the subfamily Peloropeodinae, and is considered close to the genera Micromorphus and Peloropeodes. Brief information on the habitat of M. erasmusorum is provided; in general, representatives of the new genus seem to prefer mesic riparian environments. A key to Afrotropical peloropeodine and allied genera of long-legged flies is compiled, and characters of the new genus are discussed.

INTRODUCTION
The 'World catalog of Dolichopodidae' (Yang et al. 2006) lists 15 genera in the subfamily Peloropeodinae, which is still poorly defined (Grichanov 2000). Several unrevised genera of uncertain taxonomic position included in the subfamily blur its border with Sympycninae. There are two relatively well-defined and distinctive genera (Peloropeodes Wheeler, 1890 andMicromorphus Mik, 1878) that are distributed worldwide, including the Afrotropics. These genera differ from other members of the subfamily, first of all, in having sessile hypopygia.
A male and a female of a peculiar peloropeodine species have been collected using a Malaise trap at the Sanyati Nature Farm near Loewsburg, KwaZulu-Natal, South Africa. A study of published descriptions (Parent 1938;Robinson & Vockeroth 1981;Meuffels & Grootaert 1987, 1997a, b, 2004Bickel 1992Bickel , 1999Bickel , 2004Runyon & Hurley 2003;Negrobov 2003;Grootaert 2006, etc.) has been unsuccessful in seeking an appropriate genus for this species. Subsequent treatment of collections from the Natal Musem led to discovery of two more specimens of the same species, and of a series consisting of one male accompanied by three females that represent another close species. Both species belong to a new genus that shares features of Micromorphus and Peloropeodes, but their inclusion in either of the latter genera would diffuse the generic concepts of both taxa. This paper offers a description of a new genus along with a key to all Afrotropical peloropeodine and sympycnine genera, with three species groups of Sympycnus and two genera previously included into the subfamily Peloropeodinae.

MATERIAL AND METHODS
The left and right lateral views of the hypopygium, or male genital capsule, are illustrated for the new species. In describing the hypopygium, 'dorsal' and 'ventral' refer to the morphological position prior to rotation and flexion. Thus, in figures showing a lateral view of the hypopygium, the top of the page is morphologically ventral, while the bottom is dorsal. Morphological terminology follows Robinson and Vockeroth (1981), Stuckenberg (1999), and Grichanov (2007).
All studied material is housed in the collection of the Natal Museum, Pietermaritzburg, South Africa (NMSA). TAXONOMY Subfamily Peloropeodinae Robinson, 1970 Genus Meuffelsia Grichanov, gen. n. Etymology: The genus is named for the famous Dutch researcher of the Dolichopodidae, Henk Meuffels. Gender feminine. Type species: Meuffelsia erasmusorum Grichanov, sp. n. Diagnosis: The generic diagnosis is based on the two very close included species, and lists features considered to be of generic importance.
Length less than 2.0 mm; body dark, with dark setae; dorsal part of postcranium slightly concave; face without setae, relatively broad, slightly narrowed downward; pedicel globular; postpedicel small, subtriangular; stylus dorsoapical; labella with 6 pseudotracheae; posterior part of mesonotum distinctly flattened and slightly depressed; acrostichals biserial; 6 dorsocentrals; scutellum with 2 strong bristles and 2 minute adjacent lateral hairs; fore and mid coxae with anterior and apical cilia; hind coxa with  Grichanov, sp. n., general habitus of the holotype in alcohol before dissection. 1 seta at middle; legs simple, with simple setae; mid and hind femora with strong anterior subapical seta; hind tarsus simple; wing nearly as long as body, relatively broad; m-cu short; segment 7 small, with tergum broad and sternum reduced; segment 8 large; hypopygial foramen left lateral; hypopygium with rounded-ovate cercus; hypandrium long and thick, asymmetrical, fused at base to epandrium; ventral surface of epandrium bare; surstyli asymmetrical, with left dorsal arm shorter or longer than right one, both broad, bearing a few short setae, and ventral arms of surstyli subequal in length, thin, directed ventrad, bearing a few short setae at apex; oviscapt with tergum 9+10 split medially into two arcuate narrow hemitergites, each bearing 4 short black acanthophorites; female cercus short, widened distad; anal plate broad, wider than long. Grichanov, sp. n. Figs 1-6 Etymology: The species is named in honour of Thea and Cobus Erasmus, on whose property the types were collected. Description: Colour (dry paratypes): Frons and face weakly shining bronze, brownish pollinose. Palpus and proboscis blackish brown; thorax bluish black; legs with coxae, femora and tarsi from apex of basitarsus mainly light brown; other podomeres mainly reddish yellow; abdomen black with brown sternites. Male. Head: Frons and face with black ground colour. One strong but short postvertical seta positioned far from postocular setal row; postocular setae all dark; 1 or 2 upper and 1 lower setae slightly longer than the others. Face under antennae 2× as wide as height of postpedicel. Antenna black, slightly longer than head height. Scape small, vase-like, with projecting inner angle forming distal denticle; pedicel larger, globular, with ring of short setulae and elongate apicodorsal seta; postpedicel subtriangular, as long as high at base, densely haired; stylus inserted dorsally at 2/3 from postpedicel base, 2× as long as main segments of antenna combined, shortly pubescent, with short thick segment 1 and filiform segment 2. Ratio of lengths of scape to pedicel to postpedicel to stylomere 1 to stylomere 2, 3:3:4:1:20. Palpus and proboscis yellow-brown, palpus subtriangular, with dark pubescence and dark short bristle. Thorax: With dark bristles, mesonotum blackish brown, pleura dark brown. One long and 1 short intra-alar, 1 humeral, 1 supra-alar, 2 notopleurals, 1 sutural, 1 presutural; 6 (2+4) dorsocentrals, with first slightly shorter and last slightly longer than others; 5th seta not shifted towards lateral margin; 6 or 7 pairs of biserial acrostichals extending to well-developed posterior mesonotal flattening. Upper propleuron without setae; proepisternum with 1 strong seta and 1 short hair above fore coxa. Legs: Mainly dirty yellow; mid and hind coxae, hind femur and apical segments of all tarsi distinctly brownish. Fore and mid coxae with dark anterior and apical cilia; hind coxa with 1 black bristle at middle. Fore leg without extraordinary setae; fore femur with just 1 or 2 short posteroventrals at apex; fore tibia simple, without strong setae; fore tarsus simple, with ordinary setulae. Length ratio of fore femur to tibia to tarsus (segments from first to fifth), 19:23:10:4:3:3:4. Mid femur simple, bearing 1 strong anterior subapical bristle and 1 fine posteroventral seta; mid tibia simple, with 1 very strong anterodorsal and 1 fine posterodorsal setae at basal third and 3 apical setae; no ventral setae; mid tarsus simple; segments 1-4 each with circlet of short apical spinules. Length ratio of mid femur to tibia to tarsus (segments from first to fifth), 30:31:14:5:4:3:4. Hind femur with 1 strong anterior subapical seta; hind tibia simple, with 1 strong anterior at basal 1/3, and 3 short posterodorsal, 2 or 3 very short ventral, 3 simple short apical setae, with only anterior apical seta somewhat longer, without strong dorsal subapical seta; hind tarsus simple; hind basitarsus with 1 short basoventral and 1 or 2 short apicoventral setae, with very small basal tooth posteriorly directed towards apex of hind tibia. Length ratio of hind femur to tibia to tarsus (segments from first to fifth), 35:35:15:14:6:4:5.  Habitat: The type specimens from Sanyati Nature Farm were collected in the primarily indigenous riverine bush (Fig. 7) As reported by Dr R.M. Miller, the collecting site of the other two paratypes was most probably along the small river that runs down the valley with indigenous forest. Grichanov,sp. n. Figs 8,9 Etymology: The species is named after J. Manning, who collected the type series. Description: Male. Similar to M. erasmusorum sp. n. in all respects except as noted.
Epandrium globular, asymmetrical, distinctly (1.5×) longer than high, as wide as high; hypopygium with rounded-ovate densely haired cercus; hypandrium mostly concealed, asymmetrical, slightly shifted towards left side, deeply bifurcated from its midlength (ventral view), with broad, apically pointed lobes almost equal in length and shape; aedeagus expanded at apex, with adjacent long thin process; ventral surface of epandrium bare; distal epandrial lobes short and broad, bare, without projections; surstyli

9
Comparison: The two described species can hardly be distinguished by external morphology. Nevertheless, wing vein m-cu is located slightly closer to the wing base in M. erasmusorum than in M. manningi; therefore, wing vein ratios differ somewhat in both males and females of the two species. The species are easily distinguished by hypopygium morphology. The hypandrium is deeply bifurcated, with almost equal lobes in M. manningi, but with only a large triangular lobe on the right side of the main arm in M. erasmusorum. The surstyli are rather distinct in their shape and setation pattern, and their ventral arm in M. manningi is peculiar in bearing several long and strong pedunculate setae on the basal half. The latter species also has a somewhat more elongated epandrium and smaller cercus than M. erasmusorum. It is also worth noting that the left dorsal arm is shorter than the right one in M. erasmusorum, and vice versa in M. manningi.
Key to Afrotropical peloropeodine and allied genera and species groups Regarding Afrotropical Peloropeodinae and Sympycninae, the 'World catalog of Dolichopodidae' includes 11 genera, some of which should be excluded from the list (Grichanov 1998(Grichanov , 2008a. This key does not reflect phylogenetic relations of included taxa, being compiled for practical use only. Species groups in the genus Sympycnus follow Grichanov (2008b). 11 Two rather than one postverticals; strong ventral subapical seta on hind tibia; wing veins R 4+5 and M 1+2 slightly diverging rather than parallel; strongly oblique crossvein m-cu forming acute (ca 60°) angle with CuA 1 ; mid femur with ventral bristles in basal part; male wing costa with long and thick stigma beyond R 1 ; epandrial foramen mostly middorsal . The following character states places Meuffelsia in the Peloropeodinae (see Yang et al. 2006): Occiput convex backward. Vertex nearly flat. Upper occiput slightly concave, verticals located nearly at level of lateral ocellus; 1 short postvertical. Postocular bristles onerowed. Eyes with tiny hairs. Male eyes separated on face; male face distinctly narrowing downward. Mesonotum with flat mid-posterior slope. Propleuron sparsely haired, with 1 bristle on lower portion. Mid and hind femora with 1 anterior preapical bristle. Hind coxa with 1 outer bristle at middle. Anal cell present; anal vein present as a short fold. Male abdominal tergite 7 short triangular, without hairs and bristles; sternite 7 reduced. Male genitalia relatively large and mostly exposed; surstylus well developed; epandrial lateral lobe distinct; hypandrium long.
The following character states are common to Meuffelsia, Afrotropical Micromorphus and Peloropeodes (after Grichanov 2000): Antenna slightly longer than head height. Scape small, bare, vase-like, with slightly projecting inner apex. Stylus dorsal. Palpus and proboscis short, haired; palpus with one black seta. Pseudotracheae symmetrically sclerotised. One strong vertical seta, half as long as head height; 2 strong ocellar setae of the same length with additional pair of short hairs posteriorly. Postocular setae in one row. Mesonotum distinctly flattened in front of scutellum. Mesonotum with 1 humeral, 1 posthumeral, 2 notopleural, 1 presutural, 1 sutural, 2 intra-alar and 1 supra-alar setae, 1 strong propleural seta. Scutellum with 2 strong setae, with or without very short lateral hairs. Fore tibia without major setae. Hind coxa with 1 black external seta. Mid and hind femora with 1 anterior subapical seta. Mid tarsomeres 1-4 with short apicoventral spinules. Wing costa with 2 strong bristles before humeral crossvein; upper seta longer than lower. Anal lobe long and narrow; anal vein fold-like; alula undeveloped; anal angle obtuse or absent.
Meuffelsia shares with Micromorphus such characters as simple legs, no strong dorsal seta on antennal pedicel, and distal part of CuA 1 being 4-5 times longer than m-cu. Nevertheless, Micromorphus shows striking differences to Meuffelsia: no acrostichals and basal denticle on hind basitarsus; dorsal and ventral subapical setae on hind tibia, 1 strong and 1 hair-like intra-alar setae; setose epandrial lobes at base and at apex of ventral epandrial surface; and symmetrical surstyli. Meuffelsia shares with examined Afrotropical and European species of Peloropeodes such characters as: presence of two rows of acrostichals; well developed segment 7 in males; and usually asymmetrical hypopygium. Nevertheless, Peloropeodes differs in many morphological characters: antennal pedicel with strong dorsal seta; postpedicel usually much larger than pedicel; male mid coxa with apical spine of glued cilia; hind tibia with 1 stronger subapical dorsal seta, slightly longer than diameter of tibia; various podomeres, especially on fore leg, often modified, bearing long setae or cilia; and fore tarsomere 5 always modified, with enlarged asymmetrical claws.
The combination of character states is distinctive to Meuffelsia but any of them may also occur in other Peloropeodinae. However, the postabdomen morphology is very remarkable for the genus, and the unusual male surstylus shape has not yet been described in other genera of the subfamily.


extfieldsofstudy: Biology Medicine

text:
Genotypic resistance testing in HIV by arrayed primer extension

The analysis of mutations that are associated with the occurrence of drug resistance is important for monitoring the antiretroviral therapy of patients infected with human immunodeficiency virus (HIV). Here, we describe the establishment and successful application of Arrayed Primer Extension (APEX) for genotypic resistance testing in HIV as a rapid and economical alternative to standard sequencing. The assay is based on an array of oligonucleotide primers that are immobilised via their 5′-ends. Upon hybridisation of template DNA, a primer extension reaction is performed in the presence of the four dideoxynucleotides, each labelled with a distinct fluorophore. The inserted label immediately indicates the sequence at the respective position. Any mutation changes the colour pattern. We designed a microarray for the analysis of 26 and 33 codons in the HIV protease and reverse transcriptase, respectively, which are of special interest with respect to drug resistance. The enormous genome variability of HIV represents a big challenge for genotypic resistance tests, which include a hybridisation step, both in terms of specificity and probe numbers. The use of degenerated oligonucleotides resulted in a significant reduction in the number of primers needed. For validation, DNA of 94 and 48 patients that exhibited resistance to inhibitors of HIV protease and reverse transcriptase, respectively, were analysed. The validation included HIV subtype B, prevalent in industrialised countries, as well as non-subtype B samples that are more common elsewhere. Electronic supplementary material The online version of this article (doi:10.1007/s00216-007-1775-0) contains supplementary material, which is available to authorized users.

Introduction
AIDS-acquired immunodeficiency syndrome-was first reported in 1981 and has since become a worldwide pandemic. Currently some 40 million people are HIVinfected (http://www.who.int/). Antiviral treatment is based on the use of drugs which inhibit HIV reverse transcriptase (RT) and protease (PR). The emergence of resistant viruses is a major obstacle to the long-term effective and successful therapy of HIV-infected patients. Antiviral drug resistance is caused by the presence of mutations in the PR and RT coding regions of the virus that reduce drug susceptibility compared to wild-type viruses (see e.g. [1]). Drug resistance can be determined by genotypic tests that identify resistance-related mutations in the viral genome [2]. Therefore, genotypic resistance testing has become an increasingly important process when monitoring the antiretroviral therapy of patients infected with HIV [3][4][5][6][7].
HIV genotypic resistance data are mostly obtained during the clinical routine by dideoxy sequencing [8] which is a timeconsuming process. Alternative methods for genotypic resistance testing are needed. Therefore, several different approaches have been described in the last few years, based on hybridisation [9], primer extension [10] or ligation methods [11]. Because of the tremendous variability of the viral genome, the two last approaches were restricted to a few selected mutations, however. In consequence, primary mutations were assayed but secondary mutations were not included. Primary mutations are directly responsible for resistance. Secondary or accessory mutations often cause resistance when present in combination with other mutations or compensate for the diminished replicate activity that can be associated with drug resistance [2]. The assay reported by Gonzalez et al. [9] covered a wide range of mutations based on the microarray technology developed by Affymetrix. Even with the large number of oligonucleotides that can be placed on this chip, however, it is difficult to achieve clinically reliable results, since the analysis is based merely on hybridisation discrimination and is therefore prone to being obscured by the high degree of sequence variance in HIV.
Here, we show the development and validation of a costeffective and rapid HIV resistance test that allows the detection of the mutations in PR and RT that are reported as being resistance-related by the Stanford HIV database http://hivdb. stanford.edu/). It is based on the Arrayed Primer Extension method (APEX; [12]), a process used to detect single nucleotide polymorphisms in a target DNA. By virtue of the combination of the discriminative effect of the hybridisation process and the base-pairing specificity of the DNApolymerase used for the extension reaction, the overall accuracy of the reaction can be increased 10-to 100-fold compared to merely hybridisation-based assays [13,14]. Template-dependent extension of the arrayed primers in the presence of the four different dideoxynucleotides, each labelled with a particular fluorophore, yields sequence information. Mutations can be detected by a change in the signal colour at the respective array position. In order to minimise the number of oligonucleotides while at the same time being able to analyse all relevant mutations in PR and RT, the applicability of degenerated oligonucleotides was also studied. A primer is called degenerated if several bases are possible at some of its sequence positions. Degenerated oligonucleotides have been found to be useful in PCR [15] and hybridisation assays (e.g. [16]), for example. The final objective was an adaptation of the APEX technology to the challenges provided by the high mutability of the HIV genome and the development of a quick, efficient and inexpensive genotypic test system for high-throughput clinical applications.

Sample collection
To evaluate the APEX-based genotypic resistance assay, 94 and 48 HIV-1 positive clinical samples from patients that exhibited resistance to inhibitors of HIV protease and reverse transcriptase, respectively, were analysed. The patientderived PCR-products were chosen randomly from routine clinical testing at the Department of Virology, University of Heidelberg. For all PCR products, the sequences were determined by Sanger sequencing using the ViroSeq genotyping kit (Abbott, Wiesbaden, Germany). Phylogenetic analysis showed that 76% (71/94) of the PR samples and 87% (41/48) samples of the RT region belonged to HIV-1 subtype B. The non-subtype B samples consisted of A, C, F1, CRF01_AE, CRF01_AG, CRF02_AG and CRF06_cpx (Rega HIV-1 Subtyping Tool, [17]).

Template preparation
For re-amplification, 5-μl aliquots of the original PCR-products were used as template. The 50 μl reaction mix consisted of 0.25 mM dNTPs, 0.3 mM each of forward and reverse primer, 1.25 U Thermoprime Plus DNA polymerase and reaction buffer (ABgene, Hamburg, Germany). 15% of the dTTP was replaced with dUTP in order to introduce dUTP randomly. For amplification, primers d(GGGAAGATCTGGCCTTCCTA CAA) and d(GGGCCATCCATTCGTGGC) were used for PR and d(CACCTGTCAACATAATTGGA) and d(ACTGTC CATTTATCAGGATG) for the RT region. The PCR primers were synthesised by biomers (Ulm, Germany).
PCR began with an initial denaturation step at 94°C for 2 min followed by 30 cycles at 94°C for 30 s, 56°C for 30 s and 72°C for 2.5 min, and a final extension at 72°C for 10 min. PTC-200 ThermoCyclers (MJ Research, Waltham MA, USA) were used for all amplifications. The expected fragment lengths of 559 bp (PR) and 780 bp (RT) were checked on 2% agarose gels. One hundred microlitres of PCR mix were concentrated and purified with a QIAquick PCR Purification Kit (Qiagen, Hilden, Germany), as recommended by the manufacturer, and eluted in 30 μl H 2 O.
Finally, 20 μl of each PCR product was fragmented by incubation with 0.1 U uracil-N-glycosylase in a reaction buffer of 50 mM Tris/HCl pH 9, 20 mM (NH 4 ) 2 SO 4 ) at 37°C for 1 h. The enzyme was deactivated at 95°C for 10 min, and an aliquot of digested DNA was again checked on an agrose gel.

Production of the oligonucleotide microarrays
All oligonucleotides were purchased from biomers (Ulm, Germany) or MWG (Ebersberg, Germany). Each one was 50 nucleotides in length, containing a C6-aminolinker and a 20-25mer oligo-dT spacer at the 5′-end. Attachment to epoxysilane-coated glass slides (Schott Nexterion Slide E, Mainz, Germany) occurred via the 5′-C6-aminolinker to ensure the accessibility of the 3′-terminus for the enzymatic reaction. The final spotting solution contained 50 μM oligonucleotide in 0.05 M sodium carbonate, 0.1 M sodium citrate, pH 9.5, with 3 M betaine.
In addition to the HIV-specific primers, two kinds of controls were included in the chip layout. First, 25-mer oligonucleotides that carried at their 3′-end the fluorescence labels Cy3, Cy5, TexasRed or Fluorescein, respectively, were utilised as positional controls (5′-TGGGTATAAACGATTTCGTAGATAAdye). A mixture of the four differently labelled oligonucleotides (12.5 μM each) was spotted onto the microarray. Second, to control the primer extension reaction an oligonucleotide was applied that forms a hairpin structure (NACGACCGACTT T A G G T C C C T C A A T T T T T G A G G G A C C T A AAGTCGGTCGT), thus acting as both primer and template. In analogy to the molecules that represent HIV sequences, all controls had a C6 aminolinker and an oligo-dT spacer at their 5′-ends.
For microarray production, the SDDC-2 Chip Writer contact printing robot (Engineering Services, Toronto, ON, Canada) was used. Equipped with SMP 3-pins (TeleChem, Sunnyvale, CA, USA), each contact delivered a volume of approximately 1 nl. All oligonucleotides were spotted in duplicate. After spotting, slides were incubated at 60°C for 1 h and stored at room temperature for at least 24 h.

Arrayed primer extension
Directly before an APEX reaction was performed, the microarrays were washed twice in H 2 O at 95°C to remove residual spotting solution. To block the surface, slides were incubated in 100 mM NaOH for 10 min at room temperature and washed again in H 2 O at 95°C. The primer extension was carried out directly on the slide surface in a reaction chamber formed by a 20 μl frame-seal (ABgene, Hamburg, Germany).
As estimated from a comparison to a 100 bp DNA marker ladder (MBI Fermentas, St. Leon-Roth, Germany), 600-800 ng of PCR product were used in each APEX reaction. The 20-μl reaction mix contained 10 μl of fragmented PCR product, 4 U ThermoSequenase DNA polymerase (Amersham, Freiburg, Germany), ThermoSequenase reaction buffer (Amersham) and 1.25 μM each of ddATP-TexasRed, ddCTP-Cy3, ddGTP-Fluorescein and ddUTP-Cy5 (Perkin Elmer, Boston, MA, USA). The mix of buffer, labelled ddNTPs and target DNA were first denatured for 10 min at 95°C before adding the DNA polymerase. The mix was subsequently loaded onto the microarray, and the reaction chamber was covered with a plastic film.
The slides were placed into a ThermoCycler PTC-200, TwinTower (MJ Research, Waltham, MA, USA). Temperature profiles were used as shown in Table 1. After the primer extension reaction, the slides were immediately washed in water at 95°C, for 3 min in 0.3% Alconox (Alconox, White Plains, NY, USA), again in water at 95°C, and finally in 30 mM NaCl, 3 mM Na 3 citrate (pH 8.5) in order to obtain optimal pH conditions for all four fluorescent dyes. Slides were dried in an air flow.

Signal analysis
Signal detection was performed by confocal four-colour laser scanners (ScanArray 4000XL and ScanArray 5000, Perkin Elmer, Boston, MA, USA). Intensities were measured using GenePix 4.0 quantification software (Axon Instruments, Union City, CA, USA). The data were normalised based on the quantum yields of the four fluorescent dyes and different hybridisation properties of the oligonucleotides using a software tool developed in-house. The output files presented a base call for each nucleotide position and allowed a direct comparison with the reference sequences obtained by Sanger sequencing.

Optimisation of APEX conditions
The application of APEX for HIV resistance testing required some modifications to the reaction conditions as described by Kurg et al. [12] for standard genotyping. Each oligoucleotide had a 20-25mer portion of oligo-d(T) at its 5′-end to reduce steric hindrance effects. Spotting the oligonucleotides in sodium carbonate buffer (pH 9) produced the best signal-to-background ratios. The addition of 3 M betaine to the spotting solution improved spot homogeneity [18]. Furthermore, in contrast to the temperature profile described by Kurg et al. [12], primer extension was not carried out as a one-step reaction at 48°C. At least three steps were needed for denaturation, hybridisation and specific primer extension in order to obtain significant Ramping of the in situ cycler occurred at 1°C / s signal intensities at the PR oligonucleotides (Table 1). Dissociation temperatures were even more variable for the RT oligonucleotide primers. Therefore a multi-step gradient profile was established (Table 1).

Degenerated oligonucleotide primers
Because of its replication cycle, HIV shows immense sequence heterogeneity [19][20][21][22]. To effectively analyse a wide range of samples, it is necessary to represent this very high sequence variety on the microarray. In order to reduce the number of oligonucleotides needed, the effects of wobble positions (a mixture of bases at the particular nucleotide) and substitution with inosine was investigated. The most polymorphic region of the PR gene was used as a model. It is located around codon 71. In total, 13 positions of the 20-mer primer that was used to query the nucleotide at sequence position 211 are not conserved. A probe representing the wildtype sequence was used as a control. In derivative molecules, more and more variable positions were replaced by degenerate bases, starting either from the 5′-end or 3′-end (Table 2). In one oligonucleotide (W13), all variable positions were degenerated. Oligonucleotide extension was performed at the conditions described in the "Materials and methods" section. Using a wild-type DNA-template, Cy3-ddCTP was incorporated. As shown in Fig. 1, increasing the number of degenerated YAYYARYACTGWRCYYDYDR 13 Degenerated positions are marked in red. Abbreviations are used according to the IUB code positions within the primer sequence led to a decrease in the signal. Oligonucleotides with 1-5 wobble positions at the 5′terminus exhibited reduced but still significant signal intensities compared to the unmodified probe (Fig. 1a). In contrast, the introduction of even one wobbling base at the 3′-end (3W1) reduced signal intensity dramatically. No significant signal could be detected with oligonucleotides that had more than one degenerated position near the 3′-end (Fig. 1b). Additional experiments with inosine and other sequences produced similar results (data not shown). Even though inosine should base-pair with all four bases, although to a reduced degree, its introduction close to the 3′-end resulted in a complete loss of signal. Based on these results, the oligonucleotides for the microarray were designed in such a way that no more than four wobble nucleotides or inosines were introduced as close to the 5′-terminus as possible. This strategy allowed an~16-fold reduction in the number of primers needed on the chip for genotypic testing of all major resistance sites in HIV.   Fig. 1a,b Impact of degenerated primers on APEX. Wobble sequences were introduced into the wild-type sequence at 13 positions of variable bases of a 20-mer primer starting from the 5′-end (a) or the 3′-end (b), as shown in  database http://hiv-web.lanl.gov/content/index). All sequence variations described as resistance-related and present in more than 5% or 10% of the cases of resistance to inhibitors of PR or RT, respectively, were covered by oligonucleotides on the chip. In total 368 PR-and 523 RTspecific oligonucleotides were spotted in duplicate onto the microarray (for sequences see the "Electronic Supplementary Information"). Fluorescently labelled oligonucleotides were used for positional control. Additionally, oligonucleotides that are able to self-extend by forming hairpin structures were introduced into the chip layout as a template-independent control for the primer extension  reaction; they represent template and primer at the same time (for sequences see "Materials and methods"). Figure 2 shows typical results from an APEX reaction on the PR oligonucleotides. The microarray was scanned at the wavelengths correlating to the four fluorescence dyes attached to the four dideoxynucleotides.
Initial validation of the APEX assay in comparison to standard sequencing In the analysis, patient samples were investigated that had been obtained from ongoing routine testing at the Department of Virology of the University of Heidelberg. The analysis was approved by the local ethics committees at the University of Heidelberg, and informed consent was obtained from all patients. The set used for validation was randomly chosen and independent of subtype, drug regimens or the presence of special genotypes. A total of 7,332 relevant nucleotide positions were analysed by APEX for the HIV PR region (78 nucleotides × 94 patient samples) and 4,752 for the HIV RT gene (99 nucleotides × 48 patient samples) and compared with the results obtained by standard Sanger sequencing as a reference method. For the PR gene, 92% (6,750 nucleotides) of the sequence information was in full agreement, while 6% (443) did not match entirely. About 2.5% (165) of the conflicting positions were due to unassigned nucleotides. For codons 10 and 36, for example, detection efficiency was low. Weak signal intensities were defined as (false) negatives, since it was impossible to determine the sequence with the stringent filter criteria applied. Another 2% (139) of the positions partially matched in APEX and sequencing since they yielded more than one base. Nucleotide mixtures that are only detected in the APEX assay but not in standard sequencing could occur because of the higher sensitivity of the APEX method to the presence of two bases at one site. The analysis software for standard sequencing often suppresses the weaker signal of  The different polymorphisms are shown as black dots. Each red square represents a patient sample. The majority of samples fall into two clusters which are highly associated with particular groups of mutations the two. Different results between APEX and Sanger sequencing were definitely obtained, mostly for secondary mutations, especially codons 63 and 71 in the protease. These positions are located within the most polymorphic region; more specific rather than degenerated primers are needed at these sites. Concordance for the investigated RT samples was 86% (4,099), partial concordance 4% (161) and discordance 10% (492). A portion of 5% (234) of the positions identified as different were false negatives. Figure 3 shows the degree of concordance between APEX and Sanger sequencing for the individual 26 codons in the PR gene and 33 codons of the RT gene.

Phylogenetic analysis of RT and PR sequences
At least nine different genetic HIV-1 subtypes and several recombinant forms exist [23]. Since subtype B is the predominant subtype in highly industrialised countries, most HIV-1 sequence data in databases is subtype B, even though subtype B is responsible for only 12% of global infections [24]. Since sequences from the Los Alamos HIV database were used as basis for the chip design, the microarray currently represents mainly subtype B sequences. Validation of our APEX genotyping assay was done with subtype B as well as non-subtype B samples. A phylogenetic analysis according to De Oliveira et al. [17] of the PR and RT samples used in our chip validation process showed that 71 of 94 (PR) and 41 of 48 (RT) sequences belonged to subtype B. The others consisted of subtypes A, C, F1 and recombinant A/G and A/E viruses. Table 3 shows a comparison of the results for subtype B and non-subtype B samples. Focusing only on subtype B samples, concordance values of 94% and 88% were obtained for PR and RT, respectively. In comparison, the values for non-subtype B samples were 88% and 84%. Although the concordance of the APEX results with the reference method was a little lower for nonsubtype B samples, the oligonucleotide set selected purely on subtype B sequences produced a base calling of sufficient accuracy to be used for non-subtype B samples.

Improvement in APEX detection
In general, the quality of the results from an APEX reaction depends on the functioning of the relevant primers. It is well documented that particular sequences may not perform well for various reasons. A promising and simple strategy for solving APEX detection problems is a modification of the microarray design by replacement and rejection of the ill-performing oligonucleotides. The analysis reported above was based on an analysis of one DNA strand only. Replacement of a primer by another oligonucleotide that queries the corresponding position on the second DNA strand is bound to improve overall detection efficiency, since an entirely different primer sequence is used, which in most cases is likely to perform well. This was demonstrated for the analysis of the PR gene. For ten poorly detected positions in codons 23, 30, 47, 60, 62, 63, 76 and 93, oligonucleotides were replaced with primers for the second DNA strand. Four samples with below-average concordance values (less than 92%) were tested again in eight repetitions on this second set of oligonucleotides. For all samples, the new base calling accuracy at the individual sites was in agreement with the sequence data. Although new primers had been designed for only ten positions, even the overall base calling accuracy increased significantly compared to the unmodified primer set (Fig. 4). While the replacement of individual primer oligonucleotides is timeconsuming using the a microarray system based on spotting as reported here, the flexibility of micromirror-controlled in situ synthesis could be utilised for this purpose [25]. In this way, an optimisation process involving an iterative cycle of probe design and experimental evaluation can be performed rather quickly and cost-efficiently.

Data analysis
The information gained from the experiments was subjected to a correspondence cluster analysis [26], which is an explorative computational method for the investigation of associations between variables, such as polymorphisms, and the individual samples, in a multidimensional space. It simultaneously displays data for two (or more) variables in a low-dimensional projection, thus revealing associations between them. Figure 5 shows the results of clustering the data from the PR analysis. The black dots represent individual polymorphisms, the red squares the individual patient samples. Co-localisation of samples in the blot is indicative of a strong association. It is apparent that the samples separate into two subgroups. Polymorphisms associated to one of these groups are plotted in the same direction. There are only a few polymorphisms which do not contribute to the discrimination of the samples, located at the center of the plot (point 0/0) or along a line that extends above or below this point. Data from more patients with good clinical annotation is required in order to identify in a statistically meaningful manner the variable that causes clear division into (at least) two subgroups.

Conclusions
This study demonstrates the successful application of APEX to genotypic drug resistance testing in HIV. Because of the use of degenerated primers, the number of sensor molecules could be kept relatively small. Nevertheless, the assay is not restricted to the detection of primary mutations in HIV. Instead, all resistance-related mutations presently known to be associated with drug resistance could be studied in a single experiment. With a slight decrease in the number of degenerated bases introduced into primers that bind to highly polymorphic sites, and by using primers for both strands, a specificity and accuracy similar to an analysis by standard Sanger sequencing can be achieved. Considering that polymorphisms in the HIV PR and RT genes are common, even among therapy-naïve patients, it could happen in rare cases that an individual primer on the microarray may not work for an individual patient-even with an optimised set of oligonucleotide primers-because of the presence of a mutation that is particular to this patient. However, given the fact that there seem to be subgroups of polymorphisms that are highly associated with each other, such an event would most likely not influence the overall outcome of the analysis. Even with the current setup, both HIV subtype B and non-subtype B samples can be analysed. Furthermore, the flexible array design allows an uncomplicated inclusion of additional oligonucleotides if necessary for this or other special applications. Another attractive aspect of the assay is that the entire procedure takes only a few hours, including data analysis. Therefore, this microarray approach allows genotypic resistance testing in a high-throughput manner with an accuracy that seems sufficient for routine clinical application.